Cloning and Overexpression of the 3-Hydroxyisobutyrate Dehydrogenase Gene from Pseudomonas putida E23.

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  • CHOWDHURY Emran Kabir
    <i>Department of Bioresources Science, Kochi University</i> Present address: <i>Department of Biochemistry, University of Dhaka</i>
  • AKAISHI Yuka
    <i>Department of Bioresources Science, Kochi University</i>
  • NAGATA Shinji
    <i>Department of Bioresources Science, Kochi University</i>
  • MISONO Haruo
    <i>Department of Bioresources Science, Kochi University</i>

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  • Cloning and Overexpression of the 3-Hydroxyisobutyrate Dehydrogenase Gene from<i>Pseudomonas putida</i>E23

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Abstract

  The structural gene for NAD+-dependent 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) from Pseudomonas putida E23 was cloned in Escherichia coli cells to obtain a large amount of the enzyme and its nucleotides were sequenced to study its structural relationship with other proteins. The gene encoded a polypeptide containing 295 amino acid residues and was in a cluster with the gene for methylmalonate semialdehyde dehydrogenase. Transformed E. coli cells overproduced 3-hydroxyisobutyrate dehydrogenase, and the recombinant enzyme was purified to homogeneity with a high yield. Lysine and asparagine residues, which are important in catalysis of the 3-hydroxyacid dehydrogenase family, are conserved in this enzyme.<br>

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